1. Technical Field
The field of the invention relates to methods and devices for conducting analytical or biological sample testing, such as, for example, for the purpose of identification of microorganisms or viruses in a test sample, and/or the susceptibility or resistance of the microorganisms or viruses to drugs. The invention is particularly related to the field of methods and devices for detection and analysis of nucleic acids present in a biological test sample.
2. Technical Review
Nucleic acid examination is a continually emerging area for test sample analysis including investigating genetic sequence and expression. Initially, a test sample from a human patient or other source is isolated and target nucleic acids in the sample are amplified to increase the number of copies to allow the test sample to be analyzed. The copies of target sequences are then hybridized to one or more complementary oligonucleotide probes in solution or in combination with a solid support incorporating complementary probes to form a hybridization complex. A detector probe may also be hybridized to the target sequences under some reactions. Detection and identification of the hybridization product is achieved when a signal is generated from the hybridization complex formed. By amplifying nucleic acid sequences, information about the presence or absence of microorganisms is obtained from the sample without resorting to culturing microorganisms.
An amplification method is described in U.S. Pat. No. 4,683,195 (Mullis) and U.S. Pat. No. 4,683,202 (Mullis), in which a polymerase chain reaction (PCR) utilizes DNA polymerase, complementary primer molecules and repeated cycles of thermal reactions to exponentially replicate target nucleic acid molecules. U.S. Pat. No. 5,792,607 (Backman) describes amplification methods referred to as ligase chain reactions (LCR). U.S. Pat. No. 5,744,311 (Fraiser); U.S. Pat. No. 5,648,211 (Fraiser) and U.S. Pat. No. 5,631,147 (Lohman), describe isothermal amplification systems based on strand displacement amplification (SDA). See also, Walker, et al., Nuc. Acids. Res. 20, 1691-1696 (1992) U.S. Pat. No. 5,437,900 (Burg) and EP 373 960 (Gingeras), describe isothermal amplification. Still other nucleic acid amplification methods are described in U.S. Pat. No. 5,399,491 (Kacian) and U.S. Pat. No. 5,409,818 (Davey). Each and all references noted above are incorporated herein by reference together with all other patents and literature references cited herein.
Hybridization techniques have been described for example, in U.S. Pat. No. 4,563,419 (Ranki) and U.S. Pat. No. 4,851,330 (Kohne) and in Dunn, et al., Cell 12, pp. 23-26 (1978) among many other publications. Various modifications to the hybridization reactions are known in the art including in solution hybridization or to capture probes on a solid support in one or more reaction steps.
Detection methods described in U.S. Pat. No. 4,486,539 (Kourlisky); U.S. Pat. No. 4,411,955 (Ward) U.S. Pat. No. 4,882,269 (Schneider) and U.S. Pat. No. 4,213,893 (Carrico), illustrate preparation of labeled detection probes for detecting specific nucleic acid sequences. Detectable labels have been conjugated, directly or indirectly through linker arms on either the base, sugar or phosphate moiety of one or more specific oligonucleotides. Labels known in the art include fluorochromes, radioisotopes, dyes, enzymes such as alkaline phosphatase, and luminescent or chemiluminescent molecules. The detector probes may bind to the amplified nucleic acid reaction products or amplicons. The amount of signal detected from the labeled detector probes after hybridization to amplicons reflects the presence or absence of amplicons and therefore of one or more selected target nucleic acid in the original test sample.
In general, amplifying known starting quantities of an internal control in a sample, then hybridizing with detector probes having an opposite sequence or sense to form a complex with the internal control amplicons and finally detecting signal generated provides one method to permit the qualitative and quantitative determination of target nucleic acids in a test sample.
Quantitative methods to determine the amount of target present in the test sample are described in U.S. Pat. No. 5,213,961 (Bunn); U.S. Pat. No. 5,476,774 (Wang); U.S. Pat. No. 5,705,365 (Ryder) and U.S. Pat. No. 5,457,027 (Nadeau).
Nucleic acid detection kits are commercially available and employ some of the above-referenced amplification, hybridization, labeling detection and quantitation techniques. For example, an HIV assay which detects amplified nucleic acid is described in U.S. Pat. No. 5,559,662 (Respess) and Gratzi, et al, J. Virol. Methods 66, pp. 269-292 (1997). Kits which achieve such amplification of HIV nucleic acids include the Chiron QUANTIPLEX Branched DNA and Organon Teknika NASBA-QT.
A test kit for M. tuberculosis is described in U.S. Pat. No. 5,643,723 (Persing) and nucleic acids for mycobacteria testing are described in U.S. Pat. No. 5,589,585 (Mabilat); U.S. Pat. No. 5,702,317 (Mabilat) and U.S. Pat. No. 5,849,901 (Mabilat).
Nucleic acid sequence analysis can be divided into two different categories: limited detection or low detection formats, and multi-detection or high detection formats. The distinction is the number of specific data signals or responses that can be obtained from the formats.
EP 0 875 584 describes an instrument that uses a low detection formats. A single type of fluorescent label bound to a detection probe can be measured to determine the presence of complementary nucleic acids in the test sample. By sequentially using this technique, the instrument has been shown to be able to measure at least two specific targets in a test sample. Alternatively, multiple labels can be measured in a screening assay simultaneously (eg; fluorescent or luminescent labels with different emission properties). However for practical reasons, the number of target analytes that can be measured in a screening assay remains less than ten. These techniques are considered included in the category of low detection formats.
The design or selection of a test panel in a low detection format will be determined in part by customer need, by disease incidence in a particular location, by the prevalence of co-infection of microorganisms and/or viruses and environmental conditions to name but a few. While low resolution or low detection formats such as nucleic acid detection kits noted above are useful in detecting the presence of a conserved or limited number of target sequences, these tests have been less effective for obtaining higher levels of data or detail about target nucleic acids which are needed for analysis of a particular variant, its drug susceptibility or presence of a mutation.
The second category of analytical methods is a multi-detection or high detection format. These methods are capable of providing more detailed information about the target amplicons. An example of this is the reverse dot-blot technique, wherein nucleic acids are hybridized to multiple complementary probes immobilized on a matrix. After staining, the multiple sections that become stained indicate the presence of complementary target(s). Another technique provides a light-directed, spatially addressable matrix or array on which is deposited numerous oligonucleotides.
Recent advances in large scale genetic analysis utilize oligonucleotides assembled as multi-detection arrays by microfabrication techniques. Synthesis and methods of these arrays are described in U.S. Pat. No. 5,700,637 (Southern); U.S. Pat. No. 5,445,934 (Fodor); U.S. Pat. No. 5,807,522 (Brown); U.S. Pat. No. 5,202,231 (Drmanac) and U.S. Pat. No. 5,837,832 (Chee). The arrays allow hybridization reactions in which the immobilized oligonucleotides are explored by labeled probes or labeled amplicons for identification of variants or mutations such as single or multiple base substitutions. In an example of an application, multi-detection arrays for detection of human immunodeficiency viruses are described in Fodor, Stephen et al., "Light-Directed, Spatially Addressable Parallel Chemical Synthesis", Science, Vol. 251 pp.767-773 (1991).
While both low and high detection formats have been used in analytical and diagnostic tests, they have different capacities. Low detection formats have the advantage of simplicity, relatively low cost and capacity to answer specific questions regarding a sample, e.g., does this sample contain nucleic acids typically from a region of the HIV virus. The high detection formats are more expensive but can provide even more data or information for analysis, e.g,. does this sample of HIV nucleic acids have mutations from the wild-type sequence, and if so, what are they? Because of their expense, multi-detection or high detection formats have not been viewed as a cost effective means for routine screening tests. Thus commercial tests typically have been provided as limited or low detection formats mainly to confirm a medical hypothesis as illustrated in FIG. 1.
In FIG. 1, a hypothesis that the patient is suffering from tuberculosis begins the testing process. A respiratory sample is obtained from a patient's sputum at step 10. The sample is processed at step 12 to prepare the organism for culture. The sample is then divided and one part used to culture the organisms. At step 14, a second portion of the sample is subjected to an Acid Fast Smear Test. If the test result from the Acid Fast Smear Test is negative, the physician must wait for the results of the culture as indicated at 16. If positive, the clinician processes the sample again, and is subjected to an amplified species specific TB test performed at 18. At step 22, an amplified Avium Intracellular test may be a third test performed on the test sample to identify the sample. If the test result at step 22 is negative, the test sample or a new second sample may be subjected to a subsequent test reaction or cultures to determine if pathogens are present in the sample.
FIG. 1 is a typical example of a low cost test known in the prior art, the Acid Fast "Smear" (step 14) is used to reject 90% of the test samples suspected of including Mycobacteria which are negative. Subsequent tests could be justified for use on the 10% of the samples that are positive, but even if test costs are low, the expense of labor and handling samples may not be justified if they must be used for all test samples.
There remains a need for instruments and automated methods that not only include but also proceed beyond limited detection screening for nucleic acid analysis. The availability of such an instrument will provide valuable information and data in, for example, identifying genetic disorders that may produce physiological effects which mimic or overlap other genetic disorders. There is also a need for an instrument system that can provide integrated test stations and data to determine the resistance of microorganisms to an available drug, for deciding appropriate therapy. Furthermore, such a system should aim for minimum manipulation by the researcher or clinician, especially those steps involving sample preparation, amplification, and detection. It should preferably be applicable as a tool to improve monitoring, data collecting, sequencing and genotyping of nucleic acids while limiting the risk of contamination between each test or step. The system should also be accurate, highly sensitive and available to the consumer at reasonable costs.